Project Summary The incidence of type 1 diabetes (T1D) is increasing annually in addition to other childhood-onset autoimmune diseases. Individuals at high genetic risk of T1D develop autoimmunity and progress to clinical disease at variable rates, indicating a key role for currently unknown environmental triggers. Without defining the environmental triggers of T1D autoimmunity, further advances in T1D disease prevention will be hindered. This proposal is inspired by recent strides in research defining the pathophysiology of autoimmunity in celiac disease, a disease that has significant genetic overlap with T1D. Abnormal increases in intestinal permeability seen in patients with celiac disease have also been demonstrated in patients with T1D and their relatives. This rise in intestinal permeability or ?leakiness?, is mediated by zonulin, a protein that increases paracellular molecular movement by opening intestinal tight-junctions. A medication that blocks the zonulin-mediated rise in intestinal permeability in patients with celiac disease has also been shown to block increases in intestinal permeability and autoimmunity in an animal model of T1D. This project is ancillary to a NIDDK DP3 study enrolling 72 first-degree relatives of patients with T1D with known T1D autoantibody levels. The overarching hypothesis driving this proposal is that increased intestinal permeability by zonulin excess contributes to development of T1D autoimmunity in susceptible individuals. Two specific aims are proposed: Aim 1 will test the hypothesis that zonulin elevation is seen in relatives of patients with T1D without celiac disease, and this increased zonulin will be more exaggerated in subjects with multiple T1D autoantibodies compared to those with 1 or no T1D autoantibodies. Baseline plasma zonulin levels will be compared between participants with no diabetes-specific autoantibodies, a single antibody, to those with multiple antibodies. Aim 2 will test the following hypothesis: zonulin levels will rise after a gluten-free mixed meal and this rise will be greater among subjects with multiple T1D antibodies and zonulin levels will decrease at the time of hypoglycemia, but less so in patients with multiple T1D autoantibodies. In order to complete Aim 2, plasma zonulin will be measured before and one hour following a gluten-free mixed meal and at the time of insulin-induced hypoglycemia to assess zonulin dynamics. The goal of this proposal is to establish whether there are progressively elevated zonulin levels coinciding with more advanced T1D autoimmune status in humans. Furthermore, if zonulin elevation precedes detection of autoimmunity in those at highest risk of T1D, it may contribute to the development of autoimmunity and can be investigated as a therapeutic target for prevention of T1D in pediatric patients.